Longitudinal in vivo metabolic labeling reveals tissue-specific mitochondrial proteome turnover rates and proteins selectively altered by parkin deficiency

Our study utilizes a longitudinal isotopic metabolic labeling approach in vivo in combination with organelle fraction proteomics to address the role of parkin in mitochondrial protein turnover in mice. The use of metabolic labeling provides a method to quantitatively determine the global changes in protein half-lives whilst simultaneously assessing protein expression. Studying two diverse mitochondrial populations, we demonstrated the median half-life of brain striatal synaptic mitochondrial proteins is significantly greater than that of hepatic mitochondrial proteins (25.7 vs. 3.5 days). Furthermore, loss of parkin resulted in an overall, albeit modest, increase in both mitochondrial protein abundance and half-life. Pathway and functional analysis of our proteomics data identified both known and novel pathways affected by loss of parkin that are consistent with its role in both mitochondrial quality control and neurodegeneration. Our study therefore adds to a growing body of evidence suggesting dependence on parkin is low for basal mitophagy in vivo and provides a foundation for the investigation of novel parkin targets.

Isolation of striatal synaptic mitochondria. Synaptic mitochondria were isolated using our previously published method 57 . Briefly, the striata, dissected from 3-4 mice, were pooled and homogenized in icecold MSHE + BSA using 10 strokes in a Dounce homogenizer. The homogenate was centrifuged for 3 min at 1300×g, the supernatant was collected, the pellet was resuspended in MSHE + BSA and the centrifugation step was repeated. The supernatants were pooled and centrifuged for 10 min at 21,000×g, and the resulting pellet was resuspended in 15% Percoll and layered on top of a 24% and 40% Percoll gradient (prepared from 100% Percoll solution). Following ultracentrifugation for 10 min at 30,700×g, the banding near the interface of the upper two gradient layers, containing synaptosomes, was collected and diluted in MSHE + BSA.
To isolate synaptic mitochondria, the synaptosomal fraction was transferred to a nitrogen cavitation vessel and the pressure was equilibrated to 900 psi for 15 min followed by depressurization to atmospheric pressure to release the synaptic mitochondria. The synaptic mitochondria suspension was added to the top of 24% Percoll and ultracentrifuged for 10 min at 30,700×g. The synaptic mitochondria pellet was resuspended in MSHE + BSA and centrifuged for 10 min at 8000×g. The pellet was washed twice with MAS and centrifuged for 10 min at 8000×g. The synaptic mitochondria pellet was lysed in 100 mM Tris-HCl with 4% (w/v) SDS and 0.1 M DTT adjusted to pH 7.6 using brief sonication and incubation at 95 °C for 5 min. The Pierce 660 nm Protein Assay was used for protein quantification.
Protein digestion. Mouse liver and striatal synaptic mitochondrial protein sample lysates were digested with trypsin using the filter-aided sample preparation (FASP) method 58 . Oasis mixed-mode weak cation exchange cartridges were used to desalt the resultant peptides prior to dehydration with a Savant ISS 110 Speed-Vac concentrator. Dehydrated peptides were resuspended in 0.1% formic acid prior to quantification using a NanoDrop 2000 UV-vis Spectrophotometer in conjunction with the Scopes 59 method for peptide quantification by absorbance at 205 nm.
Mass spectrometry acquisition. Untargeted proteomics of liver mitochondria. Liquid chromatography. Peptides were cleaned with PepClean C18 spin columns, re-suspended in 2% acetonitrile (ACN) and 0.1% formic acid (FA), and 2 µg of each sample was loaded onto a trap column Acclaim PepMap 100 75 µm × 2 cm C18 LC Columns at a flow rate of 4 µl/min followed by separation with a Thermo RSLC Ultimate 3000 on a Thermo Easy-Spray PepMap RSLC C18 75 µm × 50 cm C-18 2 mm column utilizing a step gradient of 9-25% solvent B (0.1% FA in 80% ACN) from 10 to 100 min and 25-45% solvent B for 100-130 min at 300 nL/min and 50 °C with a 155 min total run time.
Mass spectrometry. Eluted peptides were analyzed by a Thermo Orbitrap Fusion Lumos Tribrid mass spectrometer in data dependent acquisition (DDA) mode. Full-scan survey mass spectra were acquired in the Orbitrap at a resolution of 60,000 from 375 to 1500 m/z. The AGC target for MS1 was set at 100% and ion filling time at 50 ms. The most intense ions with charge state 2 thru 5, isolated in 3 s cycles and fragmented using HCD fragmentation at 30% normalized collision energy, were detected at a mass resolution of 15,000. The AGC target for MS2 was set at 100% and ion filling time at 22 ms with a dynamic exclusion of 15 s and a 10 ppm mass window.
Untargeted proteomics of striatal synaptic mitochondria. Liquid chromatography. Synaptic mitochondrial peptides were sent to Bioproximity and mass spectrometry was performed on a Q-Exactive HF-X Quadrupole Orbitrap instrument coupled with an EASY nLC 1200.
Mass spectrometry. Eluted peptides were analyzed by a Q-Exactive HF-X Quadrupole Orbitrap mass spectrometer in DDA mode. Full-scan survey mass spectra were acquired in the Orbitrap at a resolution of 60,000 from 350 to 1400 m/z. The AGC target for MS1 was set at 1e5 and ion filling time at 45 ms. The most intense ions with charge state 2 thru 5, isolated in 1.3 s cycles and fragmented using HCD fragmentation at 27% normalized collision energy, were detected at a mass resolution of 15,000. The AGC target for MS2 was set at 4.50e3 and ion filling time at 22 ms with a dynamic exclusion of 30 s and a 10 ppm mass window.

Mass spectrometry analysis (Abundance). Liver (n = 24; Lumos Tribrid-see Mass Spectrometry
Acquisition section) or synaptic mitochondria (n = 12; Q-Exactive-see Mass Spectrometry Acquisition section) from 3-month-old WT and PKO male mice were isolated, lysed and quantified by Pierce 660 nm protein assay. Fifty (50) µg of protein was prepared for mass spectroscopy using the FASP method as previously described 57,58 . Two (2) µg of eluted peptide from each was injected for analysis as described above. Raw files were searched using MaxQuant (2.0.3.0) 60,61 against the Uniprot Mus musculus proteome (UP000000589) using the MaxQuant 60,61 internal contaminant list to obtain protein identifications. Enzymatic cleavage specificity was set to trypsin with a maximum of two missed cleavages. Label multiplicity was set to two with a heavy label to account for tri-deuterated leucine (+ 3.0188325 Da). Variable modifications were set to oxidation (M), phospho (STY) and acetyl (N-term) and the only fixed modification used was carbamidomethyl C. Quantitation was www.nature.com/scientificreports/ achieved using the MaxQuant 60,61 standard label-free quantification algorithm with default settings plus match between runs, second peptide and dependent peptide search with an FDR of 0.01. Total LFQ intensity for each protein ID in each sample of the proteinGroups.txt file was determined by summing of light and heavy LFQintensity values prior to subsequent analysis. Using LFQ-Analyst 62 , the corrected raw data files were normalized based on the assumption that the majority of proteins do not change between the different conditions. Statistical analysis was performed using an in-house generated R script based on the proteinGroups.txt file. Contaminants, reverse hits, and proteins identified "only by site" were filtered out. Additionally, proteins identified by a single peptide and those not identified/quantified consistently in same experimental condition were also removed. The LFQ data was converted to log 2 scale, samples were grouped by conditions and missing values were imputed using the 'Missing not At Random' (MNAR) method, employing random sampling of a left-shifted Gaussian distribution of 1.8 StDev apart with a width of 0.3. Protein-wise linear models combined with empirical Bayes statistics were used for the differential expression analyses. The density of initial search results is illustrated in Supplementary Fig. 2. A list of DE proteins for each comparison was generated by the R Bioconductor package, limma 63 . An adjusted p-value cutoff of 0.05 (Benjamini-Hochberg method) and an absolute log 2 fold change of 1 were set to identify significant DE proteins in each pairwise comparison. Proteins deemed significant by this method were further subjected to a missing value cutoff, wherein only proteins that were quantified in a minimum of 50% of samples from each genotype (i.e. in hepatic and synaptic mitochondria 12 and 6 valid values, respectively, were required in each WT and PKO). Differentially expressed proteins that were quantified in both tissues were hierarchically clustered by Euclidean distances with average linkage in Perseus (1.6.15.0) 64 with 21 row clusters and all other default settings. These clustered proteins were then filtered for those that were deemed significant in one or both tissues by quantitative analysis. ]-leucine and methionine oxidation, respectively. The precursor mass tolerance was set to 10 ppm with tryptic enzyme specificity allowing two missed cleavages per peptide. False discovery metrics were re-scored with the Crux-Percolator (3.02.0) 69,70 using reversed sequences as decoys. Proteins were filtered with an FDR q-value threshold of 0.01, and the output was transformed to the BlibBuild 71 spectrum sequence list format for import into Topograph 72 using a custom R script. Protein half-lives were calculated based on data from all peptides detected using the Topograph-daily x64 release. Proteins were represented by at least 10 total values of percent newly synthesized per genotype. Shared peptides were excluded from the analysis and only unique peptides were used. Cutoffs were applied utilizing the quality controls generated by Topograph; turnover score ≥ 0.98, deconvolution score ≥ 0.95, total area under the curve ≥ 1,000,000, and data points above or below the protein mean by more than 2 standard deviations. For the half-life calculations, data points from all biological replicates were pooled. Proteins with excessive variability of percent newly synthesized values were excluded and determined as follows. To create a measure analogous to a coefficient of variation, the 95% confidence interval generated by Topograph for each half-life was divided by the half-life value. Proteins with a 95% confidence interval/half-life ratio ≥ 0.3 were excluded from the analysis 23 . This data censor resulted in a total of 742 and 412 proteins with quantified half-lives for comparison between WT and PKO mice in hepatic and striatal synaptic mitochondria, respectively ( Supplementary Fig. 3). Pathway analysis. Log 2 fold change values for all identified proteins shared by both liver and synaptic mitochondria were analyzed with the use of QIAGEN IPA (QIAGEN Inc.) 73 to identify pathways affected by parkin insufficiency. Summary networks were created for each tissue type alone, and a comparison analysis was employed to identify predicted activation of canonical pathways. Canonical pathway activation z-scores were subjected to hierarchical clustering by Euclidean distances with average linkage using Perseus (1.6.15.0) 64 , with 10 row clusters and all other default settings.

Mass spectrometry analysis (Turnover
Western blot analysis. Liver mitochondria were isolated as above and lysed using 100 mM Tris-HCl with 4% (w/v) SDS and 0.1 M DTT adjusted to pH 7.6 by sonication and incubation at 95 °C for 5 min. Protein concentration was determined using the Pierce 660 nm Protein Assay. Fifteen (15) µg of total protein was resolved on either (Nnt, ubiquitin) 4-12% Bis-Tris NuPAGE Bolt gels using a MOPS/SDS buffer system or (NdufV2) 15% Tris gels using the Tris/Glycine buffer system. Gels were transferred to poly-vinylidene-fluoride membrane using the iBlot dry transfer system. Membranes were blocked for 1 h at room temp with 3% non-fat milk in tris-buffered saline with 0.1% Tween-20 (TBST). Membranes were incubated overnight with primary antibodies diluted in 1% non-fat milk in TBST (Nnt 1:1000, Sigma Aldrich [HPA004829]; NdufV2 1:750, Sigma Aldrich [HPA004829]; total ubiquitin 1:500, Invitrogen ). Membranes were washed 3 × 10 min with TBST and then incubated with appropriate near-infrared conjugate secondary antibodies for 1 h at room temp (goat antimouse-680, goat anti-rabbit-800 1:20,000 each, Licor). After 3 additional 10 min washes with TBST membranes were imaged on a Licor Odyssey at 2 min per channel and quantified by densitometry. Expression of target protein was normalized to total protein loading via Coomassie staining and statistical analysis was performed in GraphPad Prism 9. Raw uncropped blot and Coomassie images have been provided in Supplementary Fig. 4

Ethical approval
The animal study was reviewed and approved by the University of Nebraska Medical Center Institutional Animal Care and Use Committee.

Loss of parkin alters expression of the mitochondrial proteomes of liver and synapses. First,
to assess if loss of parkin alters the abundance of mitochondrial proteins, we assessed the proteome of mitochondria from liver and brain, two organs known to show high expression of parkin 74,75 . For the brain, to increase the relevance of our work to Parkinson's disease (PD), synaptic mitochondria were isolated from the striatum, the target of projections from the SN. Expression profiles of the proteomes of mitochondria isolated from liver tissue and striatal synapses were determined by quantitative label-free proteomics. We reproducibly quantified 809 and 847 proteins in total from hepatic and synaptic mitochondrial preparations, respectively. Using an expression absolute log 2  Label-free quantitative proteomics were used to determine the effects of loss of parkin on the mitochondrial proteomes of (a) liver and (b) synaptic mitochondria. Proteins considered significantly up-(dark red) or down-regulated (dark blue) were determined by a log 2 fold change >|1| in PKO vs WT, adjusted p value < 0.05, and were quantifiable in no less than half of the samples in each genotype. Lighter colors indicate those proteins that had were quantifiable in less than half of the samples in each genotype. (c) Heatmap of DE proteins shared by liver and synaptic mitochondria and were deemed significant in liver mitochondria (black text), synaptic mitochondria (blue text), or both tissues (red text). Heatmap was generated in GraphPad Prism 9 using filtered LFQ-analyst 62  www.nature.com/scientificreports/ played similar expression directionality is both tissues. In validation of our MS1-based proteomics quantification, we immunoblotted for Nnt and NdufV2 expression in liver mitochondrial isolates (Fig. 1d,e). In support of our proteomics results expression was decreased and increased, for Nnt and NdufV2, respectively in PKO liver mitochondria compared to WT liver mitochondria. NdfuV2, not reaching significance, likely due to decreased sensitivity of immunoblot compared to LC-MS/MS.

Pathway analysis revealed global impact of parkin insufficiency.
To uncover similarities and differences in the global effects on mitochondrial protein abundance and impact on canonical pathways by parkin insufficiency, Ingenuity pathway analysis 73 (IPA) was used to analyze the quantified proteins in common to both liver and striatal. Graphical summaries of the global IPA analysis (i.e., Canonical Pathways, Upstream Regulators, Transcriptional Regulators, etc.) for hepatic and synaptic mitochondria are presented in Fig. 2a,b, respectively. Notably, in hepatic mitochondria the central node with positive activation was peroxisome proliferator activated receptor γ (Ppargc1) coupled with an overall state of activation of pathways and regulators related to mitochondrial biogenesis and liver detoxification (Fig. 2a, Supplementary Table S3). Interestingly, the sirtuin signaling pathway, which plays a role in cell survival and stress response, was determined to have a negative activation score (Fig. 2a). In synaptic mitochondria the primary central nodes, exhibiting positive activation, were "Cell survival" and "Cell viability" (Fig. 2b, Supplementary Table S4). Sirtuin signaling was also predicted and similar to hepatic mitochondria, the activation score was also negative but to a greater degree. Unlike liver mitochondria pathways related to mitochondrial biogenesis are less prevalent in striatal synaptic mitochondria when parkin is lost. A legend for symbols used in the IPA outputs is provided in Fig. 2c. To make a direct comparison between the hepatic and synaptic mitochondria, a comparison analysis was conducted using IPA and the activation z-scores of predicted "Canonical Pathways" are reported in the heatmap in Fig. 2d and the complete list is available in Supplementary Table S5 (canonical pathways are well-defined biochemical cascades in the cell that transduce a specific functional biological consequence). Although largely similar, hepatic and synaptic mitochondria differ in the Rho GDP-dissociation inhibitor (Arhgdia) signaling (positive activation in synaptic mitochondria) and pathways related to the TCA cycle, ketogenesis, ketolysis, as well as amyotrophic lateral sclerosis response (negative activation in synaptic mitochondria; Fig. 2d). The majority of the canonical pathways displaying positive activation scores in both hepatic and synaptic mitochondria are related to energy amino acid metabolism, cholesterol metabolism, and importantly, pathways of neurotransmitter degradation.

Proteins associated with ubiquitin-like conjugation processes are altered in hepatic mitochondria when parkin is lost. Functional annotation enrichment of hepatic mitochondrial DE proteins
using The Database for Annotation, Visualization and Integrated Discovery (DAVID) [76][77][78] revealed under parkin insufficiency, a cluster of eight proteins that are involved in ubiquitin-like conjugation processes (Fig. 3a). The complete output of the functional clustering is provided in Supplementary Table S6.
Loss of parkin causes the upregulation of proteins involved in protein repair and aggregate mitigation as signal transduction processes in synaptic mitochondria. Similar to hepatic mitochondria, we identified proteins that were altered by loss of parkin in synaptic mitochondrial isolates. Pathway analysis and annotation revealed that several of these proteins were related to mechanisms of protein aggregate mitigation, protein repair, and signal transduction mechanisms. Among those are four proteins related to protein repair and aggregate mitigation, all of which displayed significantly increased abundance upon loss of parkin (Fig. 3b). Similarly displaying increased expression under parkin insufficiency were four proteins annotated as involved in signal transduction (Fig. 3c). The complete output of functional clustering is provided in Supplementary Table S7. Network analysis using the STRING database revealed connections between several of the proteins identified as DE in synaptic mitochondrial isolates (Fig. 3d), many of which are impacted in neurological disorders.
Liver mitochondrial proteome dynamics in the context of parkin deficiency. To investigate the effects of parkin deficiency on liver mitochondrial protein turnover rates in vivo, we performed stable isotope metabolic labeling in mice with a synthetic diet containing 3 H 2 -leucine. Mice were acclimatized to the synthetic diet for 21 days and then placed on a deuterated-leucine diet at 73, 78, 83, and 87 days of age. At 90 days of age, after the 17-, 12-, 7-, or 3-days stable isotope labeling, liver mitochondria were isolated, protein lysates were trypsin digested, and the resultant peptides were quantified and used for shotgun proteomics followed by protein synthesis measurements with Topograph software 72 . The proteomic analysis of turnover rates for liver mitochondria revealed a similar distribution in protein half-lives between WT and parkin KO mice (Fig. 4a, Supplementary Table S8). The median half-life of 752 proteins in the WT liver mitochondria was 3.46 days, whereas it was 3.54 days in the PKO liver mitochondria. Of these 752 proteins, 449 were annotated as true mitochondrial proteins using MitoMiner, these proteins also exhibited similar distributions in both PKO and WT animals (Fig. 4b, Supplementary Table S9). The median half-life of 449 mitochondrial proteins in the WT liver was 3.79 days, whereas it was 3.82 days in the PKO liver. Upon comparison of the ratio of half-lives of each individual protein in PKO versus WT, the mean ratio of PKO/WT is greater than 1.0 by 2.2% (total proteins, Fig. 4c) and 2.8% (mitochondrial proteins, Fig. 4d). The overall percentage of proteins exhibiting a change in half-life greater than the mean ratio were 53.1% and 50.6% of total and mitochondrial proteins, respectively, suggesting at best a small overall increase in liver mitochondrial protein turnover rates with parkin deficiency. The mean log 2 ratio of PKO/WT is 0.025 (total proteins, Fig. 4e) and 0.033 (mitochondrial proteins, Fig. 4f). These changes indicate that the mitochondrial proteins in the liver have a slightly slower turnover (longer half-lives) in the absence of  Synaptic mitochondrial proteome dynamics in the context of parkin deficiency. In addition to liver mitochondria, striatal synaptic mitochondria were also isolated from the same mice as above. The proteomics analysis of turnover rates for synaptic mitochondria revealed a similar distribution in protein half-lives between WT and PKO mice (Fig. 5a, Supplementary Table S10). The median half-life of 412 proteins in the WT synaptic mitochondria was 25.8 days, whereas it was 25.7 days in the PKO synaptic mitochondria. Of these 412 proteins, 258 were annotated as mitochondrial proteins using MitoMiner, which similar to liver displayed similar distributions in WT and PKO animals (Fig. 5b, Supplementary Table S11). The median half-life of 258 synaptic mitochondrial proteins in the WT striatum was 27.0 days, whereas it was 27.2 days in the PKO synaptic mitochondria. Upon comparison of the ratio of half-lives of each individual protein in PKO versus WT, the mean ratio of PKO/WT is approximately 1.0, only lower by 0.5% (total proteins, Fig. 5c) and 0.9% (mitochondrial proteins, Fig. 5d), suggesting no change in synaptic mitochondrial protein turnover rates with parkin deficiency. The mean log 2 ratio of parkin KO/WT is − 0.011 (total proteins, Fig. 5e) and -0.017 (mitochondrial proteins, Fig. 5f). These changes indicate that the mitochondrial proteins have a similar turnover (similar halflives) in the absence of parkin.

Pathway analysis of the effect of parkin deficiency on liver mitochondrial proteome dynamics.
To identify protein pathways that are differentially regulated by parkin deficiency, we performed canonical pathway analysis of our dataset using IPA. The mitochondrial proteins sorted to 30 canonical metabolic pathways exhibiting an absolute value z-score greater than 1 and a − log(p value) greater than 1.3 (i.e. p < 0.05; Fig. 6a, Supplementary Table S12). The top pathway according to activation z-score was oxidative phosphorylation exhibiting positive activation (z-score = 5.0) based on the increased half-lives of the subunits of the electron transport chain (ETC) complexes I-V in the absence of parkin (Fig. 6b). Except for complex IV, the median halflives of the subunits for each of the ETC complexes were higher in liver mitochondria from PKO as compared to WT mice (complex V, + 0.46 > complex I, + 0.26 > complex III, + 0.25 > complex II, + 0.14 > complex IV, -0.05), www.nature.com/scientificreports/ suggesting reduced turnover (Fig. 6c). Further supporting this finding, the individual half-lives for the majority of the subunits for each of the ETC complexes were elevated in PKO mice (Fig. 6d).

Network analysis of the shortest-and longest-lived liver mitochondrial proteins in parkin KO mice.
The mitochondrial proteins exhibiting half-lives that increased (70 proteins, slower turnover) or decreased (31 proteins, faster turnover) by at least 10% in PKO mice as compared to WT mice were analyzed using the search tool for retrieval of interacting genes (STRING) to acquire protein-protein interaction (PPI) networks. Figure 7a shows the 10 significant clusters that were found in the slower turnover PPI network analysis using STRING MCL (Markov) clustering. Functional annotation of the top PPI network cluster (Fig. 7a, cluster 1) revealed enrichment of three Reactome Pathways, complex I biogenesis (MMU-6799198), respiratory electron transport (MMU-611105), and respiratory electron transport, ATP synthesis by chemiosmotic coupling, and heat production by uncoupling proteins (MMU-163200). Figure 7b shows the 3 significant clusters that were found in the faster turnover PPI network analysis using STRING MCL clustering. Functional annotation of the top PPI network cluster (Fig. 7b, cluster 1) revealed enrichment of two Reactome Pathways, mitochondrial translation elongation (MMU-5389840) and mitochondrial translation termination (MMU-5419276). Of note, while the proteins showing faster turnover are involved in mitochondrial translation, cluster 2 in Fig. 7a highlights that several ribosomal proteins showed slower turnover.
Pathway and STRING network analysis of the effect of parkin deficiency on synaptic mitochondrial proteome dynamics. Although overall there was no global effect of parkin deficiency, there were pathways that are differentially regulated by parkin deficiency that were revealed by IPA. The mitochondrial  Table S13). The top pathway according to activation z-score was "Dopamine Degradation" exhibiting positive activation (z-score = 2.0), quite interesting as the striatum is dopamine rich.

Comparison between liver and synaptic mitochondria.
Of the 449 (liver) and 258 (synaptic) proteins annotated as mitochondrial proteins using MitoMiner, 184 mitochondrial proteins were found in both datasets (Supplementary Table S14). The proteomic analysis of turnover rates for these 184 proteins revealed divergent distributions in protein half-lives between mitochondria isolated from the liver and synaptic mitochondria isolated from the striatum of WT mice (Fig. 8a, Supplementary Table S14). The median half-lives of these 184 proteins in the WT liver mitochondria were 4.03 days, whereas they were 28.6 days in the WT striatal synaptic mitochondria. Similarly, the median half-lives of the 184 proteins in the PKO liver mitochondria were 4.14 days, whereas they were 28.3 days in the PKO striatal synaptic mitochondria (Fig. 8b, Supplementary  Table S14). Thus, the mitochondrial proteins localized in the synapse have slower turnover (longer half-lives) than when found in the liver.

Proteins with altered half-lives display altered expression in hepatic and synaptic mitochondria.
Protein abundance is a balance between protein production and destruction, and alterations in half-life can affect abundance. Hepatic or synaptic mitochondrial proteins determined to be DE in abundance or with significantly altered half-lives were used in correlation analysis to determine if the change in half-life was coincident with altered expression in PKO relative to WT. These lists were comprised of 83 and 36 proteins for hepatic www.nature.com/scientificreports/ and synaptic mitochondria, respectively. We first analyzed the correlations for these proteins in each tissue, revealing Pearson's correlation coefficients (r) of 0.5907 for hepatic, and 0.5178 for synaptic. We then partitioned these lists into proteins that were directly or inversely correlated, improving the overall correlation ( Fig. 8c-f, Supplementary Table S15). Interestingly, in both hepatic and synaptic mitochondria approximately two-thirds of the proteins with altered abundance or half-life were directly correlated with the remaining one-third inversely correlated. Directly correlated proteins in liver include Nnt reduced expression and shorter half-life in PKO (implying increased destruction relative to production) and Rab7a with increased expression and half-life in PKO (implying decreased destruction relative to production). Nnt facilitates the production of NADPH from NADH and NADP, and is associated with regulating redox state 80 . Notably, Rab7a and parkin exhibit reciprocal regulation 81,82 and loss of parkin in vitro is associated with elevated Rab7 expression 82 . From synaptic mitochondria Lyrm7, a Complex III assembly factor which when mutated causes mitochondrial Complex III deficiency 83 , exhibited decreased expression and shorter half-lives. Among inversely correlated hepatic mitochondrial proteins were Atp5c1, with reduced expression and increased half-life, and Hsp90b1 with increased expression and decreased half-life. Atp5c1 encodes the gamma subunit of the catalytic core of Complex V 84 , and mutations are associated with a form of late onset AD 85 . Hsp90b1 is interesting because Hsp90 family proteins are molecular chaperones that aid in protein folding and mitigating alpha-synuclein pathology 86,87 . In synaptic mitochondria tyrosine hydroxylase, which catalyzes the rate limiting step of dopamine production, and Hspa4l, a protein that mitigates alpha-synuclein aggregation 88 , are notable for increased abundance while their half-lives are decreased. Only one protein, synaptojanin 2-binding protein (Synj2bp) exhibited inverse correlation in both. Interestingly, expression of Synj2b (also known as Omp25), which functions in recruiting cell adhesions molecules to mitochondria 89 , was elevated in both mitochondrial populations.

Discussion
The results of our study demonstrate in vivo differences in protein abundance and turnover rates within the mitochondrial proteomes derived from liver or striatal synapses, as well as tissue-specific effects of loss of parkin, offering insight into potential novel downstream targets regulated by parkin activity. Specifically, we report that loss of parkin leads to an overall general increase in protein abundance in both liver and striatal synaptic mitochondria, specifically significant differences in expression of 38 and 12 proteins, respectively. In line with this, loss of parkin in either tissue resulted in a subtle increase in overall protein half-life that was more readily apparent in hepatic mitochondrial than striatal synaptic. Importantly, overall protein half-lives were longer in striatal synaptic mitochondria (median half-life ~ 25.7 days) than of hepatic mitochondria (median half-life ~ 3.5 days). We did not find a generalized effect of parkin deficiency on protein abundance or half-life, pointing to a largely negligible role of parkin in mitochondria homeostasis, at least in the liver and striatal synaptosomes. Although it is tempting to compare protein abundance changes between hepatic and striatal synaptic mitochondria our study is limited by acquisition of raw data from mitochondria derived from each tissue using different instrumentation.

Figure 7.
In striatal synaptic mitochondria loss of parkin alters pathways related to bioenergetics, protein translation, and neurotransmitter metabolism. Network analysis of mitochondrial annotated proteins from striatal synaptic mitochondrial preparation using the STRING database that were determined to display elevated (a) or decreased (b) half-life. Clusters were determined using Markov clustering. Images in (a-b) were created using the STRING 79 database (version 11.4; https:// string-db. org) (c) IPA "Canonical Pathways" that exhibited significant activation scores. Magnitude of activation z-score is represented by color saturation gradient (i.e., the greater the magnitude the darker the color), while directionality is represented by color (i.e., orange = positive and blue = negative). Image modified from IPA output. www.nature.com/scientificreports/ Typically associated with mitochondrial damage response, the E3-ubiquitin ligase parkin is an effector of mitophagy and when mutated results in autosomal recessive juvenile PD. Historically most studies investigate parkin function and loss thereof in the context of mitophagy, which is driven in experimental systems through dramatic loss of mitochondrial membrane potential such as treatment of cells with carbonyl cyanide m-chlorophenyl hydrazone (CCCP). While an essential mitigator when mitochondrial damage occurs, mitophagy is also critical for homeostatic (i.e. basal) mitochondrial turnover aiding in the regulation of many cellular and physiological processes including cellular metabolism, calcium buffering, regulation of REDOX state, clearance of mitochondria during red blood cell maturation, neurotransmitter release, and cell survival 90 . Congruent with the importance of parkin and mitophagy in PD, we determined that parkin insufficiency resulted in changes that are associated with pathways of neurodegeneration. However, whereas mitophagy is thought to affect the mitochondrion as a whole, we only found effects on certain proteins. Specifically, loss of parkin in our study correlated with alterations in the abundance of synaptic mitochondrial proteins related to protein aggregate clearance and repair mechanisms including Hsph1, Hspa4l 88 , and Pcmt1 91 . Importantly, these have been shown to act on alpha-synuclein 88 , the major proteopathic component of Lewy Bodies which contributes to the neuropathological damage in PD 37,92 . Pcmt1 additionally functions in regulating mitochondrial morphology which is disrupted in PD 91 . Other proteins involved in myelination and cytoskeleton stability also appeared to be upregulated, perhaps as a response to myelin damage and/or improper vesicle trafficking which is seen in certain neurodegenerative (c-f) Dots represent proteins that were derived from the list of DE proteins (abundance, blue), differential halflife (turnover, black dots), or shared (both, red). Gene symbols in red represent proteins that overlap in liver and striatal synaptic lists.  34,93 . In the hepatic mitochondrial proteome, alterations in the abundance of proteins related to pathways of neurodegeneration were observed. A majority among these were multiple subunit components of respiratory Complex I, which is theorized to be to particularly susceptible to proteopathic damage 92,94,95 and is the target of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, MPTP, a neurotoxin which results in a PD-like syndrome in people 96 , and used to model PD in rodents, and its analogues 43,[97][98][99] .
To date several studies have explored the effects of parkin insufficiency on basal mitophagy in vitro (reviewed in 100 ), yet few, limited to Drosophila models, have investigated its importance under homeostatic conditions in vivo, the most prominent of which report conflicting findings 23,53 . A landmark study by Vincow et al. 23 , employing a metabolic labeling proteomics approach comparable to the one we used herein, reported both significant increases in mitochondrial protein half-lives from parkin loss of function mutant flies and selective turnover of respiratory chain components. In contrast, a more recent study suggested although basal mitophagy is plentiful in Drosophila in either larval epidermis or CNS but not adult flight wing muscle, it is only minimally impacted by loss of parkin function 53 . Notably, a key difference between these studies is the method of measuring mitophagy. Methods aside, a possible explanation for this discrepancy may be selective vulnerability in certain types and subpopulations of cells 101,102 as reported by Cackovic et al. 32 . In support of a model in which parkin serves a minor role in basal mitophagy we observed only minor elevations in the median half-lives of mitochondrial proteomes of both liver mitochondria and striatal synaptic mitochondria. In line with this, basal mitophagy was shown to occur despite the absence of parkin activation in tissues of high energy consumption including brains of PINK1 KO mice 103 , which may be the result of an alternate pathway regulated by Mul1 28,29 . Furthermore additional PINK1/parkin independent pathways that may function under homeostatic conditions have also been described 104,105 . The presence of these alternate pathways has been theorized to contribute to the poor reflection of rodent PD models in the neuropathological phenotypes observed in patients bearing loss of function mutations in either PINK1 or parkin 48,106 . Although we did not directly investigate this, we did observe elevated abundance of proteins involved in ubiquitin-like conjugation processes in hepatic mitochondrial proteomes, yet further investigation would be required to determine mechanistically if alternative pathways are acting and to what degree in this system. Together with only modest changes observed in protein abundance and turnover rates under parkin insufficiency our study lends support to recent reports suggesting that basal mitophagy is largely a parkin independent process 53,103,104,107 . With our intentional focus on mitochondrial isolates, our study does not comprehensively explore the extra-mitochondrial functions of parkin, which could be the subject of future investigations. Furthermore, although the use of tri-deuterated leucine is an established method of metabolic labeling, a limitation of this is the presence of peptides in the analysis that do not contain leucine and thus while they contribute to the expression analysis do not contribute to half-life determination.
One particularly interesting aspect of our study is the observation of tissue-specific protein turnover timescales between liver and striatal synaptic mitochondria, such that synaptic mitochondria exhibited a greater lifespan. A likely explanation for this difference is the need for mitochondria to travel long distances from the soma to the synaptic terminals where they function 108,109 . Importantly, this may also in part explain why synaptic mitochondria are particularly susceptible to damage which in turn leads to synaptic failure described is neurodegeneration 109 . Our findings are supported by earlier studies in Drosophila reporting differential mitochondrial turnover rates in different fly tissues 52 and in distinct cell types during autophagic flux in C. elegans 54 . This suggests that tissue specific regulation of basal mitophagy which may result from differential expression of upstream regulators. Our observation of altered sirtuin signaling predicted in hepatic mitochondrial samples by IPA supports this. Sirtuin signaling is central regulator of mitochondrial homeostasis contributing to mitophagy, fission fusion dynamics, and biogenesis via several pathways 110,111 . Our results may also suggest a feedback mechanism since sirtuin signaling displayed a negative activation score under conditions of parkin insufficiency, however, this was not directly addressed. In addition to sirtuin signaling, other potential regulators may also influence the differences in tissue specific mitochondrial turnover rates. One notable candidate of recent interest is the membrane associated ring-CH-type finger 5 (Marchf5, also known as MITOL, a mitochondrial resident E3 ubiquitin ligase) 21 which when depleted results in altered mitochondrial structure and promotes oxidative stress 112 . Canonical mitophagy relies on a positive feedback loop between parkin and PINK1, and MITOL was recently implicated as a potential initiation seed for recruiting parkin in a PINK1 dependent manner 21 . Although detected in our proteomics, our study design did not afford direct comparisons between tissue types under normal or parkin deficient conditions to assess if differential expression of MITOL is between hepatic and striatal synaptic mitochondria may contribute to tissue specific turnover rates. Further studies combining parkin insufficiency and mitochondrial stress will be of particular interest to further characterize tissue specific roles of parkin.
In summary our study revealed both differential basal turnover rates of hepatic and synaptic mitochondrial proteins as well as differential effects of loss of parkin. Our findings suggest that parkin function makes only modest contributions to basal mitophagy in vivo, and this is more pronounced in liver than in striatal synapses. Specifically, we report the median half-life of the synaptic mitochondrial proteome is substantially longer than that of hepatic mitochondria, a finding that supports the importance of synaptic mitochondria in neurodegenerative disorders. Notably, loss of parkin was associated with general increased protein abundance that correlated with reduced turnover (increased protein half-life). However, these changes were minor and support a recent paradigm shift in the field of mitophagy lending support to a model in which parkin-dependent mitophagy plays a modest role in basal mitophagy, suggesting additional studies should focus efforts on studying the effects of parkin insufficiency in the context of mitochondrial stress or damage. Finally, our study identified potential novel targets of parkin activity, opening new avenues for continued investigation into mechanisms of action for parkin.

Data availability
The mass spectrometry proteomics data presented herein have been deposited to the ProteomeXchange Consortium via the PRIDE 113 partner repository with the dataset identifier PXD037940 (abundance) and PXD037993 (turnover).